I'm interested if there is a way to degrade the plasmid DNA inside an E.Coli cell specifically so that the method does no harm to the chromosomal DNA. First I was thinking about restriction endonucleases but I'm afraid that;

  • it isn't specific enough and kills the cell
  • or it does nothing, because the E.Coli methylates the plasmid DNA too

My second thought was the CRISPR/Cas9 system but it sounds like an overcomplication of the problem. Any simpler ideas? (For eg. specific enzymes)

EDIT: the main idea is to create more competent E.Coli cells with directed evolution. Currently this is just a thought experiment, so there is no specific plasmid sequence. In details; I would transform the plasmid into the cells, then do the selection part with an antibiotic and let them grow, then transfer the cells into a medium where there is no antibiotic and add IPTG to induce the plasmid-DNA-hydrolase enzyme, then maybe an other selection part could be performed to remove the cells which still contain the plasmid (for example with FACS if there is a GFP gene on the plasmid), then make the cells competent again with CaCl2 and do this whole process over and over again.

  • $\begingroup$ There are a lot of (simple) solutions, but I'm not really sure what you want to achieve. Can you clarify? $\endgroup$ – VonBeche Oct 27 '16 at 8:24
  • $\begingroup$ Why do you want to do this? To get rid of the plasmid and re-use the cells for something else? $\endgroup$ – Chris Oct 27 '16 at 8:30
  • $\begingroup$ @Chris Exactly, the core idea was that if such an enzyme exists then I could elevate the competitive ability of the cells by directed evolution. $\endgroup$ – fazekaszs Oct 27 '16 at 10:11
  • $\begingroup$ Do you know the sequence of the plasmid? It would not be straightforward to get rid of the plasmid especially if it is high-copy number. $\endgroup$ – WYSIWYG Oct 27 '16 at 17:12
  • $\begingroup$ @VonBeche I tried to clarify it in the question details. What are those simple solutions? $\endgroup$ – fazekaszs Oct 28 '16 at 7:51

traditional method to remove a plasmid from E. Coli is to grow the cells with Etidium bromide.



Some of the information contained in this post requires additional references. Please edit to add citations to reliable sources that support the assertions made here. Unsourced material may be disputed or deleted.

  • $\begingroup$ Done. But really... this is like needing to reference plasmid extraction by alkaline lysis or pouring an agarose gel. $\endgroup$ – JayCkat Dec 19 '16 at 5:29

Your Answer

By clicking “Post Your Answer”, you agree to our terms of service, privacy policy and cookie policy

Not the answer you're looking for? Browse other questions tagged or ask your own question.